In Escherichia coli, the amino acids L-threonine, L-isoleucine, L-lysine and L-methionine derive all or part of their carbon atoms from aspartate (aspartic acid) via the following common biosynthetic pathway (G. N. Cohen, "The common pathway to lysine, methionine and threonine," pp. 147-171 in Amino Acids: Biosynthesis and Genetic Regulation, K. M. Herrmann and R. L. Somerville, eds., Addison-Welesley Publishing Co., Inc., Reading, Mass. (1983)): ##STR1##
The first reaction of this common pathway is catalyzed by one of three distinct aspartate kinases (AK I, II, or III), each of which is encoded by a separate gene and differs from the others in the way its activity and synthesis are regulated. Aspartate kinase I, for example, is encoded by thrA, its activity is inhibited by threonine, and its synthesis is repressed by threonine and isoleucine in combination. AK II, however, is encoded by metL and its synthesis repressed by methionine (although its activity is not inhibited by methionine or by paired combinations of methionine, lysine, threonine and isoleucine (F. Falcoz-Kelly et al., Eur. J. Biochem. 8:146-152 (1969); J. C. Patte et al., Biochim. Biophys. Acta 136:245-257 (1967)). AK III is encoded by lysC and its activity and synthesis are inhibited and repressed, respectively, by lysine.
Two of the AKs, I and II, are not distinct proteins, but rather a domain of a complex enzyme that includes homoserine dehydrogenase I or II, respectively, each of which catalyzes the reduction of aspartate semialdehyde to homoserine (P. Truffa-Bachi et al., Eur. J. Biochem. 5:73-80 (12968)). Homoserine dehydrogenase I is therefore also encoded by thrA, its synthesis is repressed by threonine plus isoleucine and its activity is inhibited by threonine. HD II is similarly encoded by metL and its synthesis is repressed by methionine.
Threonine biosynthesis includes the following additional reactions: homoserine.fwdarw.homoserine phosphate.fwdarw.threonine. The phosphorylation of homoserine is catalyzed by homoserine kinase, a protein which consists of two identical 29 kDa subunits encoded for by thrB and whose activity is inhibited by threonine (B. Burr et al., J. Biochem. 62:519-526 (1976)). The final step, the complex conversion of homoserine phosphate to L-threonine is catalyzed by threonine synthase, a 47 kDa protein encoded for by thrC (C. Parsot et al., Nucleic Acids Res. 11:7331-7345 (1983)).
The thrA, thrB and thrC genes all belong to the thr operon, a single operon located at 0 minutes on the genetic map of E. coli (J. Theze and I. Saint-Girons, J. Bacteriol. 118:990-998 (1974); J. Theze et al., J. Bacteriol. 117:133-143 (1974)). These genes encode, respectively, for aspartate kinase I-homoserine dehydrogenase I, homoserine kinase and threonine synthase. Biosynthesis of these enzymes is subject to multivalent repression by threonine and isoleucine (M. Freundlich, Biochem. Biophys. Res. Commun. 10:277-282 (1963)).
A regulatory region is found upstream of the first structural gene in the thr operon and its sequence has been determined (J. F. Gardner, Proc. Natl. Acad. Sci. USA 76:1706-1710 (1979)). The thr attenuator, downstream of the transcription initiation site, contains a sequence encoding a leader peptide; this sequence includes eight threonine codons and four isoleucine codons. The thr attenuator also contains the classical mutually exclusive secondary structures which permit or prevent RNA polymerase transcription of the structural genes in the thr operon, depending on the levels of the charged threonyl- and isoleucyl-tRNAs.
Because of the problems associated with obtaining high levels of amino acid production via natural biosynthesis (e.g. repression of the thr operon by the desired product), bacterial strains have been produced having plasmids containing a thr operon with a thrA gene that encodes a feedback-resistant enzyme. With such plasmids, L-threonine has been produced on an industrial scale by fermentation processes employing a wide variety of microorganisms, such as Brevibacterium flavum, Serratia marcescens, and Escherichia coli.
For example, the E. coli strain BKIIM B-3996 (Debabov et al., U.S. Pat. No. 5,175,107), which contains the plasmid pVIC40, makes about 85 g/L in 36 hr. The host is a threonine-requiring strain because of a defective threonine synthase. In BKIIM B-3996, it is the recombinant plasmid, pVIC40, that provides the crucial enzymatic activities, i.e, a feedback-resistant AK I-HD I, homoserine kise and threonine synthase, needed for threonine biosynthesis. This plasmid also complements the host's threonine auxotrophy.
E. coli strain 294 (E. Shimizu et al., Biosci. Biotech. Biochem. 59:1095-1098 (1995)) is another example of a recombinant E. coli threonine producer. Strain 294 was constructed by cloning the thr operon of a threonine-overproducing mutant strain, E. coli K-12 (.beta.IM4) (derived from E. coli strain ATCC 21277), into plasmid pBR322, which was then introduced into the parent strain (K. Wiwa et al., Agric. Biol. Chem. 47:2329-2334 (1983)). Strain 29-4 produces about 65 g/L of L-threonine in 72 hr.
Similarly constructed recombinant strains have been made using other organisms. For example, the S. marcescens strain T2000 contains a plasmid having a thr operon which encodes a feedback-resistant thrA gene product and produces about 100 g/L of threonine in 96 hrs (M. Masuda et al., Applied Biochemistry and Biotechnology 37:255-262 (1992). All of these strains contain plasmids having multiple copies of the genes encoding the threonine biosynthetic enzymes, which allows overexpression of these enzymes. This overexpression of the plasmid-borne genes encoding threonine biosynthetic enzymes, particularly a thrA gene encoding a feedback-resistant AK I-HD I, enables these strains to produce large amounts of threonine. Other examples of plasmid-containing microorganisms are described, for example, in U.S. Pat. Nos. 4,321,325; 4,347,318; 4,371,615; 4,601,983; 4,757,009; 4,945,058; 4,946,781; 4,980,285; 5,153,123; and 5,236,831.
Plasmid-containing strains such as these, however, have problems that limit their usefulness for commercial fermentative production of amino acids. For example, a significant problem with these strains is ensuring that the integrity of the plasmid-containing strain is maintained throughout the fermentation process because of potential loss of the plasmid during cell growth and division. To avoid this problem, it is necessary to selectively eliminate plasmid-free cells during culturing, such as by employing antibiotic resistance genes on the plasmid. This solution, however, necessitates the addition of one or more antibiotics to the fermentation medium, which is not commercially practical for large scale fermentations.
Another significant problem with plasmid-containing strains is plasmid stability. High expression of enzymes whose genes are coded on the plasmid, which is necessary for commercially practical fermentative processes, often brings about plasmid instability (E. Shimizu et al., Biosci. Biotech. Biochem. 59:1095-1098 (1995)). Plasmid stability is also dependent upon factors such as cultivation temperature and the level of dissolved oxygen in the culture medium. For example, plasmid-containing strain 29-4 was more stable at lower cultivation temperatures (30.degree. C. vs. 37.degree. C.) and higher levels of dissolved oxygen (E. Shimizu et al., Biosci. Biotech. Biochem. 59:1095-1098 (1995)).
Non-plasmid containing microorganisms, while less efficacious than those described above, have also been used as threonine producers. Strains of E. coli such as H-8460, which is obtained by a series of conventional mutagenesis and selection for resistance to several metabolic analogs makes about 75 g/L of L-threonine in 70 hours (Kino et al., U.S. Pat. No. 5,474,918). Strain H-8460 does not carry a recombinant plasmid and has one copy of the threonine biosynthetic genes on the chromosome. The lower productivity of this strain compared to the plasmid-bearing strains, such as BKIIM B-3996, is believed to be due to lower enzymatic activities particularly those encoded by the thr operon) as these non-plasmid containing strains carry only a single copy of threonine biosynthetic genes. Other examples of suitable non-plasmid containing microorganisms are described, for example, in U.S. Pat. Nos. 5,376,538; 5,342,766; 5,264,353; 5,217,883; 5,188,949; 5,164,307; 5,098,835; 5,087,566; 5,077,207; 5,019,503; 5,017,483; 4,996,147; 4,463,094; 4,452,890; 3,732,144; 3,711,375; 3,684,654; 3,684,653; 3,647,628; 3,622,453; 3,582,471; 3,580,810; 3,984,830; and 3,375,173.
In both the non-plasmid and plasmid containing strains of E. coli, the thr operon is controlled by the particular strain's respective native threonine promoter. As described above, the expression of the native promoter is regulated by an attenuation mechanism controlled by a region of DNA which encodes a leader peptide and contains a number of threonine and isoleucine codons. This region is translated by a ribosome which senses the levels of threoninyl-tRNA and isoleucinyl-tRNA. When these levels are sufficient for the leader peptide to be translated, transcription is prematurely terminated, but when the levels are insufficient for the leader peptide to be translated, transcription is not terminated and the entire operon is transcribed, which, following translation, results in increased production of the threonine biosynthetic enzymes. Thus, when threonyl-tRNA and/or isoleucinyl-tRNA levels are low, the thr operon is maximally transcribed and the threonine biosynthetic enzymes are maximally made.
In the E. coli threonine-producing strain BKIIM B-3996, the threonine operon in the plasmid is controlled by its native promoter. As a result, the thr operon is only maximally expressed when the strain is starved for threonine and/or isoleucine. Since starvation for threonine is not possible in a threonine-producing strain, these strains have been rendered auxotrophic for isoleucine in order to obtain a higher level of enzymatic activity.
Another way of overcoming attenuation control is to lower the level(s) of threonyl-tRNA and/or isoleucinyl-tRNA in the cell. A thrs mutant, for example, having a threonyl-tRNA synthase which exhibits a 200-fold decreased apparent affinity for threonine, results in overexpression the thr operon, presumably due to the low level of threonyl-tRNA (E. J. Johnson et al., J. Bacteriol. 129:66-70 (1977)).
In fermentation processes using these strains, however, the cells must be supplemented with isoleucine in the growth stage because of their deficient isoleucine biosynthesis. Subsequently, in the production stage, the cells are deprived of isoleucine to induce expression of the threonine biosynthetic enzymes. A major drawback, therefore, of using native threonine promoters to control expression of the threonine biosynthetic enzymes is that the cells must be supplemented with isoleucine.
The antibiotic borrelidin is also known to reduce the enzymatic activity of threonyl tRNA-synthetase, and thereby inhibit the growth of E. coli (G. Nass et al., Biochem. Biophys. Res. Commun. 34:84 (1969)). In view of this reduced activity, certain borrelidin-sensitive strains of E. coli have been employed to produce high levels of threonine (Japanese Published Patent Application No. 6752/76; U.S. Pat. No. 5,264,353). Addition of borrelidin to the culture was found to increase the yield of L-threonine. Borrelidin-sensitive strains of Brevibacterium and Corynebacterium have also been used to produce high levels of threonine (Japanese Patent No. 53-101591).
Borrelidin-resistant mutants of E. coli similarly exhibit changes in threonyl tRNA-synthestase activity. More specifically, borrelidin-resistant E. coli have been shown to exhibit one of the following features: (i) constitutively increased levels of wild-type threonyl tRNA-synthetase; (ii) structurally altered threonyl tRNA-synthetase; or (iii) some unknown cellular alteration, probably due to a membrane change (G. Nass and J. Thomale, FEBS Lett. 39:182-186 (1974)). None of these mutant strains, however, has been used for the fermentative production of L-threonine.
In view of the discussion above, there remains a need in the art for microorganism strains which efficiently produce amino acids such as threonine, but without the problems associated with the state of the art.